Immunizing composition containing mesoinosito-tri-alpha-d-mannoside



United States Patent s 142 620 IMMUNIZING coMPosITIoN CONTAINING MESOINOSITO-TRI-ALPHA-D-MANNOSIDE Hubert Bloch, Pittsburgh, Pa., assignor to Ciba Corpora- 5 tion, a corporation of Delaware No Drawing. Filed Mar. 23, 1961, Ser. No. 97,734

6 Claims. (Cl. 167-79) The present invention relates to new immunizing agents which have a protective action against TB infections.

It has been found that the immunizing effect of phosphatidyl-mesoinosito tri oz D mannosides is enhanced when adjuvants are added to them. A phosphatidylmesoinosito-tri-a-D-mannoside of the empirical formula C H O P melting at 208209 C. and having the specific optical rotation [a] =+46 (c. =1.02 in chloroform) was isolated from Bacillus Calmette Gurin (BCG). The elementary analysis revealed the following data:

O=S5.17%; 54.83% H=8.86%; 8.97% P=2.1%; 1.93%

For the empirical formula C H O P the following were calculated:

C==55.28% H=8.60% P=2.3%

It is to be assumed that each molecule contains on an average 2 fatty acids C H O which esterify a primary and secondary hydroxyl group each of glycerol. These acids are probably acids of the group consisting of:

Palmitic acid, a branched fatty acid having 17 carbon atoms,

A branched fatty acid having 19 carbon atoms, for instance tuberculostearic acid,

A normal fatty acid having 19 carbon atoms and lactobacillic acid.

The weak extinction of 220 my (6 120) excludes the presence of a:B-unsaturated fatty acids of the type of phthienic acid.

On hydrolysis with dilute alkali metal hydroxide solution, for example by keeping a solution of 500 mg. of phosphatide in 5 cc. of benzene, to which 0.5 cc. of an alcoholic solution of potassium hydroxide of strength 3,142,620 Patented July 28, 1964 Since the phosphatidyl-inosito-dirnannoside of optical rotation [a] =+34 reveals the presence of an ail- 6- bond of the two mannose radicals as well as an a-linkage between the dimannoside and the inositol, and since the phosphatidyl-inosito-trimannoside described above displays an even stronger dextrorotation, it may be assumed has been added, for 24 hours at room temperature, a

water-soluble substance is obtained that contains glycerol, inositol, phosphorus and mannose andthe analytical data of which correspond to a glycero-phosphoryl-inositotrimannoside.

The paper-chromatographic examination of a total hythat also in this compound there is an a-glycosidic bond between all three mannose radicals.

On the strength of these data the new trimannoside may be given the following formula:

ca ocoa paocon' o ca -o-P in which R and R is each a member of the group consisting of palmitic acid, a branched fatty acid having 17 carbon atoms, a branched fatty acid having 19 carbon atoms, a normal fatty acid having 19 carbon atoms and lactobacillic acid, and D is a mesoinositol radical.

The new trimannoside possesses valuable pharmacological properties. Inter alia, it has a beneficial influence upon the development and course of tubercularinfections. It is capable of protecting or prolonging the life of experimental animals infected with tubercle bacilli in the same way as inoculation does with living BCG bacilli prior to infection. As compared with the living vaccine, the compound has qualitatively the further advantage that it does not sensitize the animals to products of the tubercle bacilli, like tuberculin, to the same degree as BCG bacilli do. The compound is therefore intended to be used as a medicament, more especially for the treatment of diseases caused by M. tuberculosis, or as intermediate for the manufacture of medicaments.

The therapeutic effect of phosphatidyl-mesoinosito-trid-D-mannoside was demonstrated as follows:

METHOD (a) Protection against infection with the tubercle bacillus.The compound (phosphatidyl-mesoinosito-tria-D-mannoside) was homogenized for 2 /2 minutes in pyrogen-free, bi-distilled water using a homogenizer '(Virtis, 20,000 r.p.m.) so that an 0.5% fine suspension was formed which was further diluted to one-third and one-tenth of the concentration, as required. The individual doses of the suspensions consequently contained 1, 0.3 and 0.1 mg. per animal respectivelyj 12 healthy laboratory mice of strain OF weighing '17 to 19 grams were given an intraperitoneal injection of the compound on the 20th and l0thday before the infection. For the purpose of comparison a further group 'of mice was intraperitoneally inoculated once with living 'BCG bacilli 20 days before the infection.

The infection of the mice was carried out in the customary manner intravenously with a homogeneous culture of virulent tubercle bacilli (Mycob. the. var. ,humqnus strain Z so that 50% of the untreated mice died up to the nineteenth day of the infection.

The results of the treatment with the compound were seen from the length-of life of the individual mice, or the average length of life in the various groups as compared with the untreated control animals (cf. table).

(11) Test for sensitizing efiecL-Two albino guineapigs weighing 200 to 300 grams were given, as described above, two injections each of the compound or one in oculation with living BCG bacilli. After 3 and weeks an intracutaneous test with purified commercial tuberculin (PPD) 50 units per animal was made on the shorn skin in the customary manner.

RESULTS (cf. table) The prolongation of life produced with the small dose of 2 x 0.1 mg. and the higher doses of the compound used, as well as with the ECG inoculation, was significant (P 0.01), and in the case of the compound was on an average more than 17 days.

The test carried out on the guinea-pig in respect of cutaneous hypersensitivity was negative in the animals treated with the compound, but strongly positive in those inoculated with living BCG bacilli.

' The above shows that the compound has a pronounced protective eifect and the advantage of replacing a living vaccine by a defined compound which does not produce any hypersensitivity to tuberculin.

TABLE Immunizing effect of phosphatidyl-mesoinosito-tri-a-D- maimoside against TB infections in mice [Length of life of the individual animals and average length of life (2050)] For use as an immunizing agent the new compound is prepared in the form of a suspension in pyrogen-free water using a stabilizer. It may be intracutaneously, subcutaneously or intramuscularly injected once or repeated at intervals.

The new trimannoside is obtained when the portion insoluble in cold methanol of a degreased culture sediment from BCG if desired after having been extracted with ether or petroleum ether to remove any residual bacterial matter is repeatedly dissolved in a water-im miscible organic solvent, filtered and the resulting solution precipitated with a water-miscible organic solvent.

The culture sediment used as starting material is known. The bacilli were killed by means of phenol.

The degreasing is advantageously carried out with acetone or another watersoluble ketone. The washed and dried, degreased culture sediment is then extracted with hot methanol and filtered while still hot. The filtrate is cooled to 015 C., advantageously to 12-14 (3., and the precipitate formed is freed from impurities by extraction with ether. The sediment is then further purified by being dissolved in chloroform and filtered through a millipore filter to remove any residual bacterial matter and by repeated precipitation from a waterimmiscible organic solvent, preferably chloroform, and one or several water-miscible organic solvents, such as acetone or methanol. For example, the precipitate may be dissolved in chloroform and precipitated with methanol, the precipitate being dissolved again in chloroform and precipitated with acetone, and finally the precipitate may be once more dissolved in chloroform and precipitated with methanol.

As mentioned above, this application concerns an immunizing agent consisting of phosphatidyl-mesoinositotri-a-D-mannoside and an adjuvant.

As adjuvants there come into consideration: killed tubercle bacilli and parts thereof, for example walls of cells, extraction residues of tubercle bacilli such as are obtained, for instance, in the extraction with lipoid-solubilizing organic solvents, for example chloroform, particularly Wax D and peptides prepared therefrom, for example the heptapeptide containing diaminopimelic acid, and also purely synthetic adjuvants, for example aluminum hydroxide, higher alkylamines, for example hexacdecylamine and especially aminoalkanol esters of higher aliphatic hydroxamic acids and their acid addition salts and quaternary ammonium salts, for example stearyl-hydroxamic acid-(fl- N:N-diethyl-N-methyl-ammonium ethyl)-ester iodide or chloride, and other compounds described in Belgian Patent 578,257.

The above adjuvants alone (without the addition of the trimannoside) have no immunizing action in the quantity used. The trimannoside alone has a slighter immunizing effect.

The immunizing substances of the invention are to be used for the treatment of TB infections.

The new mixture of phosphatidyl-mesoinosito-tri-oc-D- mannoside and adjuvan-ts and the preparations containing the same are prepared in the customary manner. For parenteral administration the mixture is for example suspended in pyrogen-free water using the usual stabilizers and, if desired, other auxiliaries, for example preserving agents, such as chlorocresol or phenol, thickening agents, such as sodium carboxy-methylcellulose or methylcellulose, substances which retard the resorption, such as polyvinyl pyrrolidone, or wetting agents, for instance polyhydroxy-ethylene sorbitol mono oleate.

EXAMPLE 1 Suspension in distilled water:

Sodium carboxymethyl-cellulose of medium viscosity 0.2 Benzyl alcohol 1.0 Sodium chloride 0.9

EXAMPLE 3 Suspension in distilled water:

Percent Phosphatidyl-mesoinosito-tri-ocD-mannoside 0.05 Stearyl hydroxamic acid-(fl-NzN-d'iethyl-N-methylammonium-ethyl) -ester iodide 0.5 Sodium carboxymethybcellulose of medium viscosity 1.0 Polyvinyl pyrrolidone 5.0 Para-chloro-meta-cresol 0.3 Sodium chloride 0.9

The phosphatidyl-mesoinosito-tri-oc-D-mannoside used as starting material may be prepared as follows:

(a) 26.1 kg. of culture sediment BCG are washed with deionized Water and distilled acetone and in the moist I state stirred twice in 261 liters of deionized water on each occasion for 1 hour at 15 C. in an enamelled vessel and then filtered through a platen filter (Thermovyl filter; 7 platens; 43 cm. diameter). Filtration takes 25 and 20 minutes respectively. The washings are discarded.

The material is suction-filtered and the dry filter cake is suspended 3 times successively with 252 liters each time of frmhly distilled acetone free from residues at 12-14 C., and each fraction is filtered through a platen filter (semi-carbon filter; 7 platens; 43 cm. diameter). Filtering takes 25 minutes in each case. The combined acetonic filtrates are discarded. The filter cake is suctioned dry and then dried at about 20 C. in a vacuum of about 20 mm. Hg until its weight remains constant. The weight of the resulting dry, finely pulverulent, White residue amounts to 3.690 kg.

3.300 kg. of the Washed and dried culture sediment obtained in the first stage are immediately suspended in 221 liters of methanol distilled until no residue is left, at 55:1 C. in a Pfaudler stirring vessel, stirred for 6 hours and then filtered through a Sparkler filter (3 platens; 43 cm. diameter; semicarton insert; filtering time 15 minutes) maintained at the same temperature. The filtrate is cooled in the Pfaudler vessel overnight to 12-14 C. while cooling with water, the precipitate is filtered through a Sulzer pressure suction filter (40 cm. diameter; covered with semi-oarton/cotton/semi-carton) (filtering time 15 minutes to 3 hours), and the residue is dried under a high vacuum at 30 C. under a pressure not CH QH H exceeding 1 mm. Hg until its weight remains constant.

Yield: 101.5 grams of crude phosphatide; to free it from any residual bacterial matter it is dissolved in chloroform and filtered through a milli-pore filter (diameter: 0.45 The filtrate is evaporated to dryness, and the residue is suspended with stirring at room temperature in 1 liter of ether. After 20 hours this suspension is centrifuged, and the solid residue is dissolved in 50 parts of chloroform (volurnezvolume). The precipitate, obtained with an equal volume of me anol, is removed by centrifngation, dissolved in 20 parts of chloroform, and again precipitated with 30 parts of acetone. The precipitate is suctioned off and taken up in 20 parts of methanol. The precipitate is separated by centrifugation; it has a melting point of 208-209 C. and an optical rotation [m] =+46 (c.=1.02 in chloroform.

The elementary analysis reveals the following data:

The content of mannose (determined by the anthrone method) amounts to 38%.

The culture sediment BCG used in this example can be prepared, for instance, in the following manner: A Sauton medium infested with BCG is incubated for 17 days at 36 C. and the surface culture is then collected.

([1) 2.98 kg. of moist culture sediment BCG are washed twice with 30 liters of deionized water for 1 hour at 15 C. on each occasion and then filtered. The residue is extracted four times with 28 liters of acetone at 1214 C. with stirring and for 2 hours each time and then filtered. There are obtained 1.085 kg. of culture sediment moist with acetone (corresponding to 370 grams of dry product). The latter is suspended in 2 liters of methanol without being dried, stirred at 20 C. for half an hour and then filtered. The filter cake moist with methanol is extracted under reflux in two batches each with 2 liters of methanol three times, for example in a Soxhlet apparatus. The resulting methanol extracts are cooled for 10 hours at +5 C. The precipitate is then filtered ofl? and dried for 60 hours at 20-25 C. under 1 mm. of

o K031 OH pressure. There [are obtained 28.4 grams of dry product. 10 grams of the product are heated for 20 minutes with ml. of ether under reflux and then filtered. The residue is taken up in 70 ml. of acetone and heated for 30 minutes under reflux. Filtration is carried out again and the extraction with ether and acetone repeated twice. Finally, the residue which is insoluble in acetone is dissolved in ml. of chloroform and then precipitated with 200 cc. of acetone. The resulting product (2.5 grams) shows the same activity as that described in Example 1.

What is claimed is:

1. An immunizing agent consisting essentially of phosphatidylnnesoinosito-tri-a-D-mannoside and an adjuvant and a pharmaceuticai carrier.

2. An immunizing agent as claimed in claim 1 wherein the adjuvant is a member selected from the group consisting of killed tubercle bacilli, and cell walls thereof, Wax D from tubercle bacilli and peptides obtained from tubercle bacilli.

3. An immunizing agent consisting essentially of a phosphatidyl-mesoinosito-tri-u-D-mannoside of the formula CH OCOR cHocoR o o 0 g OH on on OK in which D is mesoinosito and in which R and R is each a member of the group consisting of palmitic acid, a branched fatty acid having 17 carbon atoms, a branched fatty acid having 19 carbon atoms, a normal fatty acid having 19 carbon atoms and lactobacillic acid, and a pharmaceutical carrier.

4. An immunizing agent as claimed in claim 3 wherein the adjuvant is a member selected from the group consisting of killed tubercle bacilli, and cell walls thereof, Wax D from tubercle bacilli and peptides obtained from tubercle bacilli.

5. An immunizing agent as claimed in claim 1 wherein the adjuvant is a member selected from the group consisting of aluminum hydroxide, higher alkylamines and lower amino laikanol esters of higher aliphatic hydroxamic acids and their therapeutically acceptable acid addition and quaternary ammonium salts.

6. An immunizing agent as claimed in claim 3 wherein the adjuvant is a member selected from the group consisting of aluminum hydroxide, higher alkylamines and lower amino alkanol esters of higher aliphatic hydroxamic acids and their therapeutically acceptable acid addition and quaternary ammonium salts.

References Cited in the file of this patent Anderson: J. Biol. Chem., note p. 618.

Anderson: J. Biol. Chem, vol. 125, 1938, pp. 299-308, note p. 299 and 308.

Suto-Nagy: J. Biol. Chem, vol. 171, pp. 749-765, note p. 760 and 761.

Myers: Adv. Tuberc. Basel, New York, 1957, Views Opposing BCG.

Report of AD Hoc Advisory Committee on BCG to the Surgeon General of the US. Public Health Service, Amer. Rev. of Tuberculosis, Pulmonary Diseases, vol. 76, pp. 726-733, 1957.

Freund: Annual Review of Microbioiogy, page 295.

Nojima: Chem. Abst., vol. 53, p. 18155 (1959).

Vilkas: Chem. Abst, vol. 55, p. 12537 (h-i), 1961, Abst. of Bull. Soc. Chem, vol. 42, pp 1013-22, 1960.

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vol. 97, 1932, pp. 617-637, 

1. AN IMMUNIZING AGENT CONSISTING ESSENTIALLY OF PHOSPHATIDYL-MESOINOSITO-TRI-A-D-MANNOSIDE AND AN ADJUVANT AND A PHARMACEUTICAL CARRIER. 